BIODIVERSITAS ISSN: 1412-033X Volume 20, Number 8, August 2019 E-ISSN: 2085-4722 Pages: 2417-2426 DOI: 10.13057/biodiv/d200841 Morpho-anatomical structure and DNA barcode of Sonchus arvensis L. DWI KUSUMA WAHYUNI 1,, SHILFIANA RAHAYU 2 , PUTUT RAKHMAD PURNAMA 1 , TRIONO BAGUS SAPUTRO 3 , SUHARYANTO 4 , NASTITI WIJAYANTI 4 , HERY PURNOBASUKI 1 1 Department of Biology, Faculty of Science and Technology, Universitas Airlangga. Kampus C, Jl. Mulyorejo, Surabaya 60115, East Java, Indonesia. Tel.: +62-31-5936501, 5924617, Fax.: +62-31-5936502, email: [email protected]2 Department of Biology, Faculty of Science and Technology, Universitas Islam Negeri Sunan Kalijaga. Jl. Adi Sucipto, Sleman 55281, Yogyakarta, Indonesia 3 Department of Biology, Faculty of Science, Institut Teknologi Sepuluh Nopember. Jl. ITS, Surabaya 60111, East Java, Indonesia 4 Faculty of Biology, Universita Gadjah Mada. Jl.Teknika Selatan, Sekip Utara, Bulaksumur, Sleman 55281, Yogyakarta, Indonesia .Email: dwi Manuscript received: 8 July 2019. Revision accepted: 31 July 2019. Abstract. Wahyuni DK, Rahayu S, Purnama PR, Saputro TB, Suharyanto, Wijayanti N, Purnobasuki H. 2019. Morpho-anatomical structure and DNA barcode of Sonchus arvensis L. Biodiversitas 20: 2417-2426. Tempuyung or show thistle (Sonchus arvensis L.) belongs to the Asteraceae. Morpho-anatomy and DNA (Deoxyribonucleic Acid) barcoding of the plant correlates with species identification and metabolite synthesis. This research aims to look at morpho-anatomical structures and analyze the DNA barcode of Sonchus arvensis L (tempuyung). Three samples used for morpho-anatomical analysis are leaves, stems, roots, fruit, and seeds. Anatomical samples are made using the embedding method. DNA barcode uses multiple locus from plastid genome: rbcL and matK. Morpho-anatomical structure of tempuyung showed a similar structure of Sonchus genus. The stem presents in the intercellular space, whereas the roots and leaves present in the vascular tissue and the seeds. Fruits present in each part of the body. Tissues that formed root are epidermis, cortex, endoderm, and stele. Tissues that formed stem are epidermis, cortex, and stele. Tissues that formed leaf are epidermis, cortex, phloem, and xylem. Tissues that formed the fruit and seed are paranormal and sclerenchyma tissues. Sonchus arvensis sequence for rbcL has a similar 100% maximum identity to rbcL gene of S. arvensis, Sonchus asper, and Sonchus oleraceus, whereas Sonchus arvensis matK sequence has a similar 99.31% maximum identity to other S. arvensis matK sequences in BLAST system. These findings provide morpho-anatomical features and DNA barcoding for identification of S. arvensis. from others species in the same genus. Thus also can be considered as pharmaceutical standard. Keywords : Anatomy, DNA barcoding, matK, morphology, Sonchus arvensis, rbcL, tempuyung INTRODUCTION Indonesia has many potentials of medicinal plants that have not been studied, at least 9,600 species of plants have medicinal properties. One of them is Sonchus arvensis L., which is found throughout Indonesia and known as an invasive plant. The local name of S. arvensis in Indonesia is tempuyung. Tempuyung belongs to the Asteraceae family and is known to have many benefits for treating asthma, bronchitis, cough, and has antibacterial, anti- inflammatory, antioxidant, diuretic, sedative, and hypnotic activity (Delyan 2016). Most of S. arvensis metabolites are contained in the leaves. It has chemical compounds such as flavonoids (kaempferol, luteolin-7-glucoside, and apigenin- 7-O-glucoside), coumarin, and taraxasterol (Sriningsih et al. 2012). Sulaksana et al. (2004) and Delyan (2016) also reported that tempuyung leaf contains high flavonoid and triterpenoids. Species genus Sonchus is distinguished among themselves by a life form, lamina shape, stem character, number of flowers in inflorescences and color, number of edges on the achenes, achenes size and color, and so on (Svitlana et al. 2018). Mejias et al. (2012) reported that Sonchus could differ by the size of flowers, stamens size, and morphology of chromosomes. Qureshi et al. (2008) reported that Sonchus genus could differ by pollen analysis, whereas anatomical analysis of genus Sonchus has not been reported. According to Sukandar and Safitri (2016), the use of tempuyung as medicine is safe even for pregnant women without side effects. Secondary metabolite compounds are located explicitly in particular part of the plant so that it may differ in cells, tissues, or organs of a plant. Secondary metabolite compounds in a plant can be detected by screening the plant extracts. Besides screening, an anatomical analysis is also crucial in determining the distribution of metabolite compounds in cells or tissues within each plant organ. In addition to morpho-anatomical studies, it is also essential to do a molecular study for identification species. Previous research from Qureshi et al. (2008) also reported about pollen morpho-anatomical studies in Sonchus genus to find different and similar characters, but in vegetative organs not done. Species identification method living thing has developed from morphological identification to molecular identification based on short DNA pieces that are called “DNA barcode” (Hebert et al. 2013). DNA barcode has applicative functions for example for the ecological survey (Dick and Kress 2009), identification taxon-taxon cryptic (Lahaye et al. 2008), and confirmation of plant samples medicines (Xue and Li 2011). Consortium for Barcode of Life (CBOL) recommends the use of two
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BI ODI VE RS I TAS ISSN: 1412-033X
Volume 20, Number 8, August 2019 E-ISSN: 2085-4722 Pages: 2417-2426 DOI: 10.13057/biodiv/d200841
Morpho-anatomical structure and DNA barcode of Sonchus arvensis L.
DWI KUSUMA WAHYUNI1,, SHILFIANA RAHAYU2, PUTUT RAKHMAD PURNAMA1,
TRIONO BAGUS SAPUTRO3, SUHARYANTO4, NASTITI WIJAYANTI4, HERY PURNOBASUKI1 1Department of Biology, Faculty of Science and Technology, Universitas Airlangga. Kampus C, Jl. Mulyorejo, Surabaya 60115, East Java, Indonesia.
Tel.: +62-31-5936501, 5924617, Fax.: +62-31-5936502, email: [email protected] 2Department of Biology, Faculty of Science and Technology, Universitas Islam Negeri Sunan Kalijaga. Jl. Adi Sucipto, Sleman 55281, Yogyakarta,
Indonesia 3Department of Biology, Faculty of Science, Institut Teknologi Sepuluh Nopember. Jl. ITS, Surabaya 60111, East Java, Indonesia
4Faculty of Biology, Universita Gadjah Mada. Jl.Teknika Selatan, Sekip Utara, Bulaksumur, Sleman 55281, Yogyakarta, Indonesia
.Email: dwi Manuscript received: 8 July 2019. Revision accepted: 31 July 2019.
Abstract. Wahyuni DK, Rahayu S, Purnama PR, Saputro TB, Suharyanto, Wijayanti N, Purnobasuki H. 2019. Morpho-anatomical structure and DNA barcode of Sonchus arvensis L. Biodiversitas 20: 2417-2426. Tempuyung or show thistle (Sonchus arvensis L.)
belongs to the Asteraceae. Morpho-anatomy and DNA (Deoxyribonucleic Acid) barcoding of the plant correlates with species
identification and metabolite synthesis. This research aims to look at morpho-anatomical structures and analyze the DNA barcode of
Sonchus arvensis L (tempuyung). Three samples used for morpho-anatomical analysis are leaves, stems, roots, fruit, and seeds. Anatomical samples are made using the embedding method. DNA barcode uses multiple locus from plastid genome: rbcL and matK.
Morpho-anatomical structure of tempuyung showed a similar structure of Sonchus genus. The stem presents in the intercellular space,
whereas the roots and leaves present in the vascular tissue and the seeds. Fruits present in each part of the body. Tissues that formed root
are epidermis, cortex, endoderm, and stele. Tissues that formed stem are epidermis, cortex, and stele. Tissues that formed leaf are epidermis, cortex, phloem, and xylem. Tissues that formed the fruit and seed are paranormal and sclerenchyma tissues. Sonchus arvensis
sequence for rbcL has a similar 100% maximum identity to rbcL gene of S. arvensis, Sonchus asper, and Sonchus oleraceus, whereas
Sonchus arvensis matK sequence has a similar 99.31% maximum identity to other S. arvensis matK sequences in BLAST system. These
findings provide morpho-anatomical features and DNA barcoding for identification of S. arvensis. from others species in the same genus. Thus also can be considered as pharmaceutical standard.
Keywords: Anatomy, DNA barcoding, matK, morphology, Sonchus arvensis, rbcL, tempuyung
INTRODUCTION
Indonesia has many potentials of medicinal plants that
have not been studied, at least 9,600 species of plants have
medicinal properties. One of them is Sonchus arvensis L.,
which is found throughout Indonesia and known as an
invasive plant. The local name of S. arvensis in Indonesia
is tempuyung. Tempuyung belongs to the Asteraceae
family and is known to have many benefits for treating
asthma, bronchitis, cough, and has antibacterial, anti-
inflammatory, antioxidant, diuretic, sedative, and hypnotic
activity (Delyan 2016). Most of S. arvensis metabolites are
contained in the leaves. It has chemical compounds such as
flavonoids (kaempferol, luteolin-7-glucoside, and apigenin-
7-O-glucoside), coumarin, and taraxasterol (Sriningsih et al. 2012). Sulaksana et al. (2004) and Delyan (2016) also
reported that tempuyung leaf contains high flavonoid and
triterpenoids.
Species genus Sonchus is distinguished among
themselves by a life form, lamina shape, stem character,
number of flowers in inflorescences and color, number of
edges on the achenes, achenes size and color, and so on
(Svitlana et al. 2018). Mejias et al. (2012) reported that
Sonchus could differ by the size of flowers, stamens size,
and morphology of chromosomes. Qureshi et al. (2008)
reported that Sonchus genus could differ by pollen analysis,
whereas anatomical analysis of genus Sonchus has not been
reported.
According to Sukandar and Safitri (2016), the use of
tempuyung as medicine is safe even for pregnant women
without side effects. Secondary metabolite compounds are
located explicitly in particular part of the plant so that it
may differ in cells, tissues, or organs of a plant. Secondary
metabolite compounds in a plant can be detected by
screening the plant extracts. Besides screening, an anatomical analysis is also crucial in determining the
distribution of metabolite compounds in cells or tissues
within each plant organ.
In addition to morpho-anatomical studies, it is also
essential to do a molecular study for identification species.
Previous research from Qureshi et al. (2008) also reported
about pollen morpho-anatomical studies in Sonchus genus
to find different and similar characters, but in vegetative
organs not done. Species identification method living thing
has developed from morphological identification to
molecular identification based on short DNA pieces that
are called “DNA barcode” (Hebert et al. 2013). DNA
barcode has applicative functions for example for the
ecological survey (Dick and Kress 2009), identification
taxon-taxon cryptic (Lahaye et al. 2008), and confirmation
of plant samples medicines (Xue and Li 2011). Consortium
for Barcode of Life (CBOL) recommends the use of two
WAHYUNI et al. – Morpho-anatomical and molecular characterization of Sonchus arvensis
2419
plastid genes, for examples rbcL and matK as barcodes
standards (Hollongsworth et al. 2009).
Genes from the plastid genome are uniparentally
inherited non-recombining, and an inherently stable
genome (Kress et al. 2005). Current results designate that
at least two plastid genes, better a multi-locus code, are
required to specify a consistent plant DNA barcode, those
are combination gene like rbcL, matK and internal spacer
such as trnH-psbA (Lucas et al. 2012). RbcL is commonly
used in phylogenetic analysis and can be simply to amplify;
the sequence can also align in nearly all terrestrial plants.
Thus rbcL is one of the right DNA barcoding regions for plants at the family and genus levels (Li et al. 2004).
So far, study about the molecular indicator of Sonchus
sp has been widely conducted by using various marker
such as ISSR (Psaroudaki et al. 2015; Subositi and Mujahid
2019), RAPD (Elkamali et al. 2010; Doğan et al. 2018),
multiple-locus barcode matK-ITS (Kim et al. 2007; Mejías
et al. 2018) moreover complete plastid genome (Cho et al.
2019; Kim et al. 2019). However, still, limited data provide
molecular marker, primarily for S. arvensis using rbcL and
matK locus.
Morpho-anatomy structures and DNA barcodes affect
the biological systems of the plant, including the
synthesizing process of secondary metabolites, so morpho-
anatomical characters and DNA barcodes contribute the
pharmaceutical standard. Because of Sonchus genus has
several plant types so that it is vital to know the morpho-
anatomical characters and DNA barcodes to prevent
misusing plants. This study aims to describe morpho-anatomical characters and to analyze DNA barcodes
(rbcL+matK) of Sonchus arvensis L.
MATERIALS AND METHODS
Plant materials The material used is the tempuyung plant (Sonchus
arvensis L.) which has been grown in Medicinal Plant
Garden “Taman Husada Graha Family”, Surabaya,
Indonesia and determined in Purwodadi Botanic Gardens,
Indonesian Institute of Sciences, Pasuruan, Indonesia.
Morpho-anatomical characterization The component observed in the morphological study is
a description of tempuyung's whole organs. The study was
conducted on three different individual plants. The
components observed in morphological studies are root,
stem, leaves, flower, and fruit. The anatomical character
studies are the cells contained in the tissue, secretion cells;
tissue contains in the organs (root, stem, leaf, fruit, and
seed). Sample preparation is using paraffin embedding with
Pollen Spheroidal Spheroidal, tricolporate Spheroidal, tetracolporate anomocytic Fruit and
seed
Hard, brown, wrinkled Hard, brown, narrow,
wrinkled
Hard, brown, most wrinkled Hard, brown, wrinkled
Branching Near inflorescence Near inflorescence From stem Near inflorescence
WAHYUNI et al. – Morpho-anatomical and molecular characterization of Sonchus arvensis
2421
Figure 1. Sonchus arvensis L. morphology, A. Vegetative phase, B. Generative phase, C. Root, D. Shoot, E. Leaf; E1. Abaxial roset leaf (RL) and inflorescence leaf (IF), E2. Adaxial RL and IF, F. Bract (Br), G. Trichome on the flower (Tr), H. Petal (Pe) and Sepal (Se), I.
Fruits arrangements in flower, J. Fruit (fr) and Pappus (pa)
Tempuyung's stem has a structure that is not much
different from other Asteraceae plants too. The external anatomical arrangement of the stem is one layer of a flat-
shaped epidermis, a single-layered of collenchyma,
irregular cortical cell shape, and more abundant than
epidermal cells (Figure 2.B). The cortex consists of 7-9
layers, usually oval-shaped or rectangular. The cortex
containing latex are seen transparently. The transport system is open collateral (xylem and phloem are limited by
cambium tissue) and arranged circularly around the pith.
The xylem trachea cells are plentiful, and wall thickened,
while the phloem is located outside the xylem and is
D
3 cm
E1
RL
IL
8 cm
E2
RL
IL
8 cm
3 cm
G
1 cm 0.5 cm
10 cm
A
10 cm
B
2 cm
C
1 cm
H
1 cm
I
Br Tr
F
Pe
fr
pa
J Se
B I O DI VERS I TAS 20 (8): 2417-2426, August 2019
2422
limited by the cambium tissue. Adult phloem cells do not
have a cell nucleus, whereas, in young phloem, the cells are
small and still have no cell lysis, thus resembling cambium
and making it difficult to distinguish. Phloem sometime
consists of amylum, its similar to other species in genus
Sonchus like S. erzincanicus (Kandemir et al. 2006).
Cambium cells are tiny and thin-walled, making it
challenging to observe. The black arrows in Figure 2.B3
indicate the elongation of the transport system to the outer
part of the cortex. This indicates the presence of leaf
forming activity. In the deepest part, there is a pith that has
a large-dense cell. There are many interstitial spaces in the cortex (Figure 2.B2) which have darker shades. Those may
be the storages of secondary metabolite secretion.
The arrangement of leaf tissue from the outside consists
of a flat, one-layered epidermis which has thickened
cuticle. There are many trichomes on the surface, but they
are not visible (Figure 2.C). Midrib has a triangle-shaped
and has 1 or 2 layers collenchyma under the epidermis. In
the midrib of the leaf (Figure 2.C1), there is a thick
irregularly shaped cortex, an open collateral transport
system, and an unclear cambium wall. Therefore it is
difficult to observe. In the mesophyll tissue section, there
are more densely colored cells. In the phloem, there is a
more concentrated interstitial space. This indicates the
presence of secondary metabolite storage in that section.
Sclerenchyma cells in bundles are dense.
The lamina (Figure 2.C2) is composed of a larger
epidermis compared to the midrib, beneath the epidermis is
a mesophyll tissue composed of the thick palisade, and spongy tissue are not distinct. The spongy tissue is spaced
apart. On the sample presented, the sponge tissue was lysis
during the preparation, so that it is difficult to observe.
There is much chlorophyll in the mesophyll. The vegetative
organ in genus Sonchus is similar so that it will work hard
to distinct species by anatomical characteristic (Subositi et
al. 2018).
The anatomical arrangement of fruits and seeds of
tempuyung is very distinctive. The tempuyung fruit is
grooved and hard, while the seeds are inside (Figure 3.D1).
In younger fruits, the seeds have not formed so that the
seed chamber is empty. Tempuyung's fruit wall is
thickened. Most of the forming tissues of tempuyung's fruit
are deep parenchymal tissues, in which there is a starch granule (shown by black arrows in Figure 3.D2) and
contains bioactive compounds. Also, there is a thickening
of sclerenchyme in the grooves of the fruit, small
sclerenchyma cells, compacted, and has a thick cell wall.
Besides strengthening, sclerenchyme tissue also serves as a
transport system on the fruit and seeds.
The middle layer is thickened with a cell structure that
extends over the seeds of the starch layer (Figure 3.D2).
The structure of the seed endosperm is very distinctive,
composed of densely tight, small meristematic cells and
many starch granules which indicates secondary metabolite
compounds therein. The endosperm of the tempuyung plant
is in two pieces, so it is considered as dicotyledons.
DNA barcoding Both rbcL and matK genes were successfully amplified
in S. arvensis. These primers were specially designed to
amplify both genes among the Asteraceae family.
Table 2. An anatomical character among Sonchus genus
Character S. arvensis S. oleraceus S. asper
Root Epidermis small thick wall and round in shape
Cortex large, polygonal and dense
color
There is air space in cortex
The transport system is radial
Stele type actynostele
Epidermis small thick wall and round in shape
Cortex large, round
The transport system is radial
Stele type actynostele
Visible cambium
Epidermis small thick wall and round in shape
Cortex polygonal
The transport system is radial
Stele type actynostele
Visible cambium
Stem One layer epidermis, flat in shape.
One layer collenchyma
7-9 layers cortex, irregular in shape
The transport system is open
collateral.
Phloem consist of starch
One layer epidermis, flat in shape.
2-3 layers collenchyma
6-7 layers cortex consist of latex
The transport system is collateral.
Phloem consist of starch
Visible cambium
One layer epidermis, rectangular.
2-3 layers collenchyma
6-7 layers cortex consist of oxalate crystals, polygonal in shape
The transport system is collateral.
Visible cambium
Leaf One layer epidermis with thick cuticula, and less trichoma,
rectangular.
Palisade and sponge tissue are not distinct.
The transport system is open
collateral
One layer epidermis with thick cuticula, and many trichomas,
rectangular.
Palisade and sponge tissue are not distinct.
The transport system is open
collateral
One layer epidermis with thick cuticula, and many trichomas,
rectangular.
Palisade and sponge tissue are not distinct.
The transport system is open
collateral.
Visible cambium.
WAHYUNI et al. – Morpho-anatomical and molecular characterization of Sonchus arvensis
2423
Figure 2. A Cross-section of roots, A1. Root base [Epidermis (Ep), Cortex (Co), Air space (AS), Phloem (Phl), Xylem (Xy)]. A2. Root
cap [Epidermis (Ep), Cortex (Co), Phloem (Phl), Xylem (Xy)]. B. Cross-section of shoots, B1. Shoot layers [Epidermis (Ep), Cortex
(Co), Air space (AS), Phloem (Phl), Xylem (Xy)], B2. Secondary metabolite accumulation (SM), B3. Transport system elongation (TSE)
The sequence length from rbcL was 433 bp, and matK
was 288 bp, whereas GC contents were 42.7% and 35.4%,
respectively. The BLAST result (Table 3) for rbcL barcode
displayed that S. arvensis MN206020 (this study) for rbcL
has a similar 100% maximum identity to rbcL gene of S.